Bacterial Staining Notes
- Staining is a commonly used microbiological technique that is used for
differentiation of microorganisms & identification of some bacterial
structures
- Staining allows for visualization of bacterial cells because it increases contrast during
microscopic imaging.
- Cellular Morphology & Cellular arrangement can be determined by observing
bacteria that have been stained through a light microscope.
- Spherical (Circular shaped in a chain) cells are arranged in a chain called
streptococci.
- Stains are commonly used for pathogen identification process, classification
of organisms, & diagnosis of disease. NOT THE TREATMENT OF DISEASE.
- Bacterial cells that are curved rods or comma-shaped are called Vibrio,
whereas helical bacteria are referred to as spirilla/spirochetes.
- Bacterial cells that are rod shaped are called BACILLI.
- To prepare a smear, a sample of bacteria is taken from a culture and spread onto a
clean microscopic slide.
- Bacterial Smears provide scientists the means to look at microorganisms.
(Preparation of bacterial smears is an integral step in the visualization of bacterial
samples).
- Preparation of bacterial smears is required for bacterial staining procedures.
(Bacterial staining procedures always begin with preparation of bacterial smears).
- Preparing smears disperses bacterial cells on a slide so that individual cells can be
visualized under the microscope. (Bacterial smears help distribute bacteria on a slide
so that individual cells can easily be visualized under the microscope).
- What is the purpose of a simple stain? To visualize bacteria & determine cell
shape and arrangement of the specimen.
- What is the purpose of the smear preparation? To allow for visualization
of individual bacteria.
- What can be determined through simple staining of a microbial smear?
Cellular arrangement & cell morphology
- Before bacteria from a solid culture are applied to a slide, a drop of sterile water is
typically added to the slide in order to: reduce the tendency of the bacteria to
stick together (the water allows the bacteria to be suspended and therefore
mare easily dispersed, & help the bacteria spread evenly
- Heating too briefly may result in bacteria not adhering to the slide (Not
heating long enough will not allow the bacteria to adhere to the slide and they may
be rinsed off during staining).
- What is the proper amount of time required for methylene blue dye to remain on the
bacterial smear during a simple stain? 30 seconds to 2 minutes.
, - Steps for preforming a smear preparation from a solid culture: 1. Label the slide
with the name of the organism. 2. Apply loopful of sterile water to slide.
3. Add a very small amount of organisms to water on slide. 4. Disperse
organisms evenly over the center of the slide. 5. Air dry. 6. Heat fix
- What is the purpose of heat fixation? Adhere the organisms to the slide &
inactivate microorganisms placed on the slide.
- Steps for performing a smear preparation from a liquid culture: 1. Label
microscope slide. 2. Apply loopful of bacterial culture to slide. 3. Air dry. 4.
Heat fix.
- Heat fixation should take place right after air drying of smeared bacterial cells
- Result of not allowing a slide to air dry sufficiently prior to heat fixation: The
bacteria may rinse off of the slide, the bacteria could be aerosolized, the
shape and arrangement of the cells may be disrupted.
- Best method for heat fixation: The heating should be very brief. Passing the
slide through the flame for 305 seconds is enough.
- Which of the following would be appropriate for choosing a sample to perform a
smear preparation? Use only a small amount of bacteria on the loop & use
bacteria from an isolated colony.
- Why is it important to air dry the bacteria before they are immobilized by heat
fixation? This ensures the optimal preservation of the bacterial morphology.
- What is the most likely reason for the poor appearance of the bacteria on this slide?
Slide was heat fixed for too long
- A smear preparation that is too light may results in: Difficulty locating cells
to observe, & inability to distinguish individual cell morphology.
- If organisms are washing off of the slide during rinsing of the stain, which steps in the
smear preparation may have been excluded? Heat fixation, air drying.
- Excessive heat fixation can cause damage to which structures, lowering the quality of
the stained preparation? Cell membrane, Capsule, Cell Wall
- What errors during the simple stain procedure could lead to an inability to visualize
bacteria on a slide? Too many bacteria were applied during the smear
preparation, stain was not applied for long enough, bacteria were not fixed
to the slide, slide was not rinsed after staining.
- The simple stain does allow for the visualization of shape & arrangement.
But NOT specific identification of a species.
- What will happen if the heat fixation step is omitted? The cells may wash off
of the slide.
- Excessive heat during the fixation stage of preparing a smear can lead to the
following outcomes upon staining: Bacterial arrangements may be disbursed &
bacterial cells may show altered morphology.
- Staining is a commonly used microbiological technique that is used for
differentiation of microorganisms & identification of some bacterial
structures
- Staining allows for visualization of bacterial cells because it increases contrast during
microscopic imaging.
- Cellular Morphology & Cellular arrangement can be determined by observing
bacteria that have been stained through a light microscope.
- Spherical (Circular shaped in a chain) cells are arranged in a chain called
streptococci.
- Stains are commonly used for pathogen identification process, classification
of organisms, & diagnosis of disease. NOT THE TREATMENT OF DISEASE.
- Bacterial cells that are curved rods or comma-shaped are called Vibrio,
whereas helical bacteria are referred to as spirilla/spirochetes.
- Bacterial cells that are rod shaped are called BACILLI.
- To prepare a smear, a sample of bacteria is taken from a culture and spread onto a
clean microscopic slide.
- Bacterial Smears provide scientists the means to look at microorganisms.
(Preparation of bacterial smears is an integral step in the visualization of bacterial
samples).
- Preparation of bacterial smears is required for bacterial staining procedures.
(Bacterial staining procedures always begin with preparation of bacterial smears).
- Preparing smears disperses bacterial cells on a slide so that individual cells can be
visualized under the microscope. (Bacterial smears help distribute bacteria on a slide
so that individual cells can easily be visualized under the microscope).
- What is the purpose of a simple stain? To visualize bacteria & determine cell
shape and arrangement of the specimen.
- What is the purpose of the smear preparation? To allow for visualization
of individual bacteria.
- What can be determined through simple staining of a microbial smear?
Cellular arrangement & cell morphology
- Before bacteria from a solid culture are applied to a slide, a drop of sterile water is
typically added to the slide in order to: reduce the tendency of the bacteria to
stick together (the water allows the bacteria to be suspended and therefore
mare easily dispersed, & help the bacteria spread evenly
- Heating too briefly may result in bacteria not adhering to the slide (Not
heating long enough will not allow the bacteria to adhere to the slide and they may
be rinsed off during staining).
- What is the proper amount of time required for methylene blue dye to remain on the
bacterial smear during a simple stain? 30 seconds to 2 minutes.
, - Steps for preforming a smear preparation from a solid culture: 1. Label the slide
with the name of the organism. 2. Apply loopful of sterile water to slide.
3. Add a very small amount of organisms to water on slide. 4. Disperse
organisms evenly over the center of the slide. 5. Air dry. 6. Heat fix
- What is the purpose of heat fixation? Adhere the organisms to the slide &
inactivate microorganisms placed on the slide.
- Steps for performing a smear preparation from a liquid culture: 1. Label
microscope slide. 2. Apply loopful of bacterial culture to slide. 3. Air dry. 4.
Heat fix.
- Heat fixation should take place right after air drying of smeared bacterial cells
- Result of not allowing a slide to air dry sufficiently prior to heat fixation: The
bacteria may rinse off of the slide, the bacteria could be aerosolized, the
shape and arrangement of the cells may be disrupted.
- Best method for heat fixation: The heating should be very brief. Passing the
slide through the flame for 305 seconds is enough.
- Which of the following would be appropriate for choosing a sample to perform a
smear preparation? Use only a small amount of bacteria on the loop & use
bacteria from an isolated colony.
- Why is it important to air dry the bacteria before they are immobilized by heat
fixation? This ensures the optimal preservation of the bacterial morphology.
- What is the most likely reason for the poor appearance of the bacteria on this slide?
Slide was heat fixed for too long
- A smear preparation that is too light may results in: Difficulty locating cells
to observe, & inability to distinguish individual cell morphology.
- If organisms are washing off of the slide during rinsing of the stain, which steps in the
smear preparation may have been excluded? Heat fixation, air drying.
- Excessive heat fixation can cause damage to which structures, lowering the quality of
the stained preparation? Cell membrane, Capsule, Cell Wall
- What errors during the simple stain procedure could lead to an inability to visualize
bacteria on a slide? Too many bacteria were applied during the smear
preparation, stain was not applied for long enough, bacteria were not fixed
to the slide, slide was not rinsed after staining.
- The simple stain does allow for the visualization of shape & arrangement.
But NOT specific identification of a species.
- What will happen if the heat fixation step is omitted? The cells may wash off
of the slide.
- Excessive heat during the fixation stage of preparing a smear can lead to the
following outcomes upon staining: Bacterial arrangements may be disbursed &
bacterial cells may show altered morphology.
